In a given living organism, thousands of genes and their products (i.e., RNA and proteins) function in a complicated and orchestrated way that creates the mystery of life. Traditional methods in molecular biology, biochemistry and cell biology generally work on a “one target (i.e., gene, protein) in one experiment” basis. This means that the throughput is very limited, and it is hard to obtain the “whole picture” of gene function and the “dynamic network” of protein function (i.e., protein-protein, protein-small molecule interaction, signaling pathways).
In the past several years, researches in the field of genomics have endeavored to develop analytical methods to simultaneous, quantitative monitor the expression levels of many thousands of genes in an organism. Similarly in the field of proteomics, the race to develop analytical methods for the simultaneous, quantitative analysis of many proteins with respect to their abundance, locations, modifications, temporal alterations, and interactions with other biological and chemical molecules has increased.
Among the various potential methods, microarray technology has been widely viewed as a revolutionary approach, which enables the simultaneous analysis of potentially thousands of molecular parameters within a single experiment. An array is an orderly arrangement of samples. Development of surface-based assays in which numerous molecules of biological interest are immobilized on a surface in a spatially addressable manner has been an important characteristic of microarray technology. Currently, biological microarrays almost all immobilize many biological molecules on the surface of a solid substrate. Each immobilized molecule is confined in a limited space on the substrate surface (i.e., a microspot with a typical diameter of several hundreds of microns), and each corresponding microspot is arranged in a defined location in a spatially addressable manner. The immobilized molecules are generally referred to as “probes,” and the corresponding microspots are called “probe microspots.” Immobilization of probe biological molecules is generally two-dimensional in nature, although the immobilization can be achieved by two different mechanisms (physical adsorption versus covalent/affinity attachment).
When the microarray is exposed to a sample of interest, molecules in the sample selectively and specifically binds to their binding partners in the microarrays. The molecules in the sample to be detected and identified are called “targets.” The binding of a “target” to the microspots occurs to an extent determined by the concentration of that “target” molecule and its affinity to a particular probe microspot. In principle, if the target concentrations are known, the binding affinity of the target to different probe microspots can be estimated simultaneously. Conversely, given the known affinities of different targets in a sample for each probe microspot, the amounts of binding observed at each microspot may be directly used to simultaneously estimate the concentrations of multiple targets in the sample. Furthermore, the pattern of binding of a target to different probe microspots can give rise to extremely useful information about the selectivity and specificity of the target to the probes in the microarrays.
To date, biological microarrays can be classified into five general categories based on the species of molecules immobilized on a surface. A first category includes DNA microarrays, which involve a set of nucleic acid molecules tethered to a surface at defined locations. The tethered nucleic acids, such as cDNAs and oligonucleotides, have known sequences and function as “probes.” The free nucleic acid sample whose identity/abundance is being assayed is the “target.” (See Nature Genetics 1999, 21(supplement), pp. 1-60.)
A second category comprises protein microarrays of various kinds. Typically, protein microarrays use immobilized protein molecules of interest on a surface at defined locations. (See review, Wilson, D. S. and Nock, S., “Functional Protein Microarrays,” Curr. Opinion in Chemical Biology 2001, 6, 81-85.) The immobilized protein molecules have known sequences and function as “probes”; whereas, a “target” is the free biological in a sample whose identity/abundance is being detected. For instance, protein microarrays have been used to identify small-molecule-binding proteins. (Zhu, H., et al., “Global Analysis of Protein Activities Using Proteome Chips,” Science 2001, 293, 1201-2105.) Arrays of antibody probes also have been used for protein profiling, to measure protein abundances in blood, to measure cytokine abundances, as well as to capture leukocytes/phenotyping leukemias. Arrays of antigen probes have been used for reverse immunoassay to measure auto-immune antibodies and allergies. When the probes are peptides, the peptide microarray may be used to measure protein kinase activities. (Houseman, B. T., et al., “Peptide chips for the quantitative evaluation of protein kinase activity,” Nature Biotechnology 2002, 20, 270-274.)
Biological membrane microarrays which require that both the receptor molecules of interest and the associated lipid membrane molecules to be immobilized on a surface in confined locations make up a third category. The immobilized receptor molecules are “probes”, whereas the “targets” are the free biologicals and chemicals in a sample whose identify/abundance is being detected. Probes can be G protein-coupled receptors (GPCRs) that are embedded in biological membranes to form GPCR arrays, which have been used to study and profile compound specificity and selectivity. (Fang, Y., Frutos, A. G., Lahiri, J., “Membrane Protein Microarrays,” J. Am. Chem. Soc. 2002, 124, 2394-2395.) Arrays with probes composed of gangliosides that are embedded in lipid membranes have been used to detect toxins in a sample. (Fang, Y., Frutos, A. G., Lahiri, J., “Ganglioside Microarrays for Toxin Detection,” Langmuir, 2003, 19, 1500-1505.)
Carbohydrate microarrays constitute a fourth category, which involves immobilized oligosaccharides as probes on a surface at defined locations. The “targets” being free in a solution sample are carbohydrate-binding proteins. The carbohydrate microarrays have been used to detect carbohydrate-protein interactions (Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W., “Oligosaccharide Microarrays for High-Throughput Detection and Specificity Assignments of Carbohydrate-Protein Interactions,” Nature Biotechnology, 2002, 20, 1011-1017.), and to identify cross-reactive molecular markers of microbes and host cells (Wang, D., Liu, S., Trummer, B. J., Deng, C., and Wang, A., “Carbohydrate Microarrays for Recognition of Cross-Reactive Molecular Markers of Microbes and Host Cells,” Nature Biotechnology, 2002, 20, 275-281.).
A fifth category includes capture reagent microarrays that involve immobilized protein-binding agents of interest on a surface at defined locations. The protein-binding agents are generated from combinatorial methods. The agents may include RNA/DNA aptamers, allosteric ribozymes, and small molecules. (See Wilson, D. S., et al., Curr. Opinion in Chemical Biology 2001, 6, 81-85.)
Assay methods to detect the binding of targets to probes in the arrays commonly measure some physical change (such as fluorescence, mass, interfacial properties, luminescence, etc.), which results from the binding of targets to probe microspots directly. These methods generally involve two-units, a probe on the array and a target to be detected in solution. The target molecules are generally labeled by a fluorescent dye. In some other cases, they used a third unit or moiety to amplify the signals. For example, an alternative method to detect targets in a sample after bound to the probe microspots is a “sandwich” assay, which employ a third unit, such as an antibody, that can bind to the target directly. The third unit is fluorescently labeled or conjugated to an enzyme that can produce fluorescence or a luminescent or colored product when supplied with the appropriate substrate.
One problem with which all current biological microarrays face is that they lack a standard by which to evaluate the array quality (i.e., probes in the arrays) as well as normalize the signals generated from the binding of targets. For example, in DNA microarrays scientists have to use a two-color hybridization technique to generate a relative differential expression pattern. Hence, a system that makes use of a universal readout is needed.